The Experimental Study Of Selective COX-2 Inhibitor Combined With Radiotherapy For Lung Cancer A549 Cells

Objective To observe the growth and apoptosis of the lung cancer A549 cell by a selective cyclooxygenase-2 inhibitor (Celecoxib) combined with radiotherapy,and conducts the function mechanism of the research, provides the theory basis for the union treatment of lung cancer.Methods (1)thiazolyl blue (MTT) colorimetric assay the effect of lung cancer A549 cell survival by different concentrations of celecoxib. (2)thiazolyl blue (MTT) colorimetric detection the effect of lung cancer A549 cells by different doses of X-ray. (3)based on (1)+(2) experimental results, select the appropriate drug concentration (celecoxib: 200 umol/L) and X-ray dose (6Gy). (4) MTT experiment: Experimental sub-groups: blank control group (PBS group), the drug group (celecoxib: 200umol/L), radiotherapy (6Gy) and the combined treatment group (divided into three groups: the concentration of celecoxib respectively 100umol/L, 200umol/L, 400umol/L, respectively together with the radiation dose 6Gy), lung cancer A549 cells were cultured on 96-hole boards after implemented these interventions, continuous observation cells for 4～6 days, testing OD value, calculated in accordance with cell growth inhibition rate (E): E = (1-average OD value of experimental group/the average OD value of control group)×100%, depicting cell growth inhibition curve. (5)laser scanning confocal (LSCM) test Apoptosis change of lung cancer A549 cell:ibid grouping, cells were cultured on 6-hole boards after the implementation of interventions, to fix cells after 48 hours, cells of the nuclear staining and laser scanning confocal microscope to observe the group morphological changes of the nucleus. (6)flow cytometry analysis (FCM) detection of cell cycle and apoptosis: ibid grouping, cells cultured in 6-hole boards after the implementation of interventions, to collect cells after 48 hours, washed by PBS, fresh specimens were detect apoptosis by staining double-Annexin-FITC-PI, while 70% ethanol fixed cells for cell cycle checkpoints. statistically analyzed each group of cells fraction in G0/G1 phase, S phase, G2/M phase and the apoptosis rate of each group cells. (7)RT-PCR detection the change of the expression of COX-2mRNA: ibid grouping, after the implementation of interventions, cells cultured in 6-hole boards, collect cells after 48 hours, washed by PBS, Trizol cracker cells, reverse transcriptase synthesis of cDNA, and use PCR for detection of the expression of COX-2mRNA of each group .Results (1)Celecoxib has the obviously growth multiplication inhibitory action to the lung cancer A549 cell, and assumes the dosage and the time dependence. (2)In a certain dose range, the X-ray has the inhibitory action to the growth of lung cancer A549 cell. (3) compared with the pure medicine group and the X-ray group, Celecoxib combines with the X-ray may strengthen the inhibitory action which obviously grows to the lung cancer A549 cell(P<0.05). (4)Celecoxib combines with the X-ray obviously promotion lung cancer A549 cell apoptosis, and we can see the formation of apoptotic bodies. (5)various experimental groups may suppress the lung cancer A549 cell growth to multiply, the union group function is more remarkable, mainly causes the cell to gather at the G0/G1 period, cells reduce at the S period and the G2/M period(P<0.05). (6)other experimental groups COX-2mRNA expression reduces except for the radiotherapy group COX-2mRNA expression increased, the COX-2mRNA expression obviously reduces in the unites group.Conclusion (1)lung cancer A549 cells significantly are inhibition of proliferation by celecoxib combined X-ray, so that the cells are gather in G0/G1 period, increase the rate of apoptosis, and we can see the formation of apoptotic bodies; (2)COX-2 inhibitor combined with radiotherapy can be reduced radiation-induced high COX-2mRNA expression, thereby inhibiting COX-2 to promote the occurrence and development of lung cancer; (3)celecoxib is a Radiosensitizer role to lung cancer A549 cells, and its mechanism may be related to COX-2mRNA expression level; (4)radiotherapy United COX-2 inhibitors may be a new approach of clinical radiotherapy synergistic.